Oncogenic herpesvirus engages the endothelial transcription factors SOX18 and PROX1 to increase viral genome copies and virus production

Abstract

27Kaposi sarcoma (KS) is a tumour of endothelial origin caused by KS herpesvirus (KSHV) infection 28 and suggested to originate from lymphatic endothelial cells (LECs). While KSHV establishes latency 29 in virtually all susceptible cell types, LECs support a spontaneous lytic gene expression program with 30 high viral genome copies and release of infectious virus. Here, we investigated the role of PROX1, 31 SOX18 and COUPTF2, drivers of lymphatic endothelial fate during embryogenesis, in this unique 32 KSHV infection program. We found that these factors were co-expressed in KS tumours with the 33 viral lytic marker K8.1, and that SOX18 and PROX1 regulate KSHV infection via two independent 34 mechanisms. SOX18 binds to the viral origins of replication and its depletion or chemical inhibition 35 significantly reduced the KSHV genome copies in LECs. PROX1 interacts with ORF50, the initiator 36 of the lytic cascade, increases lytic gene expression and virus production and its depletion reduces 37 KSHV spontaneous lytic reactivation. Upon lytic replication, PROX1 binds to the KSHV genome in 38 the promoter region of ORF50 and enhances its transactivation activity. These results demonstrate 39 the importance of two endothelial transcription factors in the regulation of the KSHV life cycle and 40 introduce SOX18 inhibition as a potential, novel therapeutic modality for KS. 41 42 43 KEYWORDS 44 Kaposi sarcoma, KSHV, herpesvirus, K8.1, transcription factors, PROX1, SOX18, lymphatic 45 endothelial cells, viral genome replication, viral reactivation. 46 47 3 MAIN 48Kaposi sarcoma (KS) is an angiogenic endothelial tumour caused by KS herpesvirus (KSHV). KS is 49 the most common cancer among AIDS patients and a frequent malignancy in Sub-Saharan Africa 1 . 50 KS presents with skin lesions that progress from patch to plaque and ultimately to nodular tumours; 51 in advanced disease visceral involvement is also seen 2,3 . The histopathological hallmark of KS is the 52 presence of KSHV-positive spindle cells (SC), the tumour cells of KS 2,3 . The cell of origin of SC has 53 been debated for two decades 3 . The prevailing hypothesis suggests lymphatic endothelial origin, 54 although blood endothelial cells or mesenchymal cells are also candidates 1,4 . In vitro, latency is the 55 default replication program in KSHV-infected cells with undetectable levels of lytic genes expressed. 56 However, KSHV infection of lymphatic, but not blood, endothelial cells (LEC and BEC) leads to a 57 unique infection program characterized by high KSHV genome copies, spontaneous lytic gene 58 expression and release of infectious virus 5-8 . 59 60 During embryonic development, LEC precursors originate from COUPTF2/SOX18 double-positive 61 BECs that physically separate from the cardinal vein to establish a primary lymphatic vascular plexus. 62 In this process, COUPTF2 and SOX18 drive the expression of PROX1 thereby orchestrating LEC 63 differentiation 9,10 . Here, we explored the role of these transcription factors (TF), instrumental for 64 establishment...